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Abstract

Introduction

Cardiovascular disease (CVD) is the leading cause of death in patients with inflammatory
polyarthritis (IP), especially in seropositive disease. In established rheumatoid
arthritis (RA), insulin resistance (IR) is increased and associated with CVD. We investigated
factors associated with IR in an inception cohort of patients with early IP.

Methods

Patients with early IP (two or more swollen joints for four or more weeks), aged 18
to 65 years, seen within 24 months of symptom onset were recruited from the Norfolk
Arthritis Register (NOAR), a primary-care-based inception cohort. Assessment included
joint examination, current and prior therapy and completion of the Health Assessment
Questionnaire. Fasting blood was taken for measurement of CVD risk factors, rheumatoid
factor (RF), anti-citrullinated protein antibodies (ACPA), C-reactive protein (CRP),
and insulin levels. IR was calculated using the homeostatic model assessment (HOMA-IR).
We examined factors associated with IR using univariate and multivariable linear regression
models.

Conclusions

Seropositivity for RF or ACPA was associated with IR in this early IP cohort. This
association may, in part, explain why seropositive patients have excess CVD mortality.

Introduction

Cardiovascular disease (CVD) remains the leading cause of death in patients with inflammatory
polyarthritis (IP) and is particularly associated with seropositive disease [1-4]. Insulin resistance (IR) is known to be increased in patients with established RA
[5,6] and has been shown to be a risk factor for both clinical CVD [7] and subclinical atherosclerosis [8-10]. It remains unclear, however, whether IR occurs early in the course of IP or whether
it develops later in the disease as a consequence of drug therapy, especially steroid
exposure, physical inactivity or changes in body habitus, such as increased body fat:muscle
ratio.

Established risk factors for IP development include smoking and obesity, both of which
are also risk factors for CVD and have been associated with IR in the general population.
It is, therefore, reasonable to consider whether IR relates primarily to these factors
rather than to the inflammatory disease process per se [11]. There is also evidence that the association of IR with established RA may be mediated
through the effects of systemic inflammation and/or glucocorticoid therapy [12]. Reduction in inflammatory biomarkers via glucocorticoid therapy, disease modifying
anti-rheumatic drugs (DMARDs), anti-tumour necrosis factor (TNF)-α therapy or weight
loss have all been associated with improvement in IR in RA [13-17]. It is, therefore, difficult to fully determine the direction of any associations
found in established RA and what the key factor(s) related to IR are in this population.

The aim of this study, therefore, was to investigate the prevalence of IR in patients
with early IP and to determine whether IR was associated with IP-related factors.
In particular we were interested in examining if IR was related to inflammatory disease
burden, serological status or early therapy exposure.

Materials and methods

Setting

The Norfolk Arthritis Register (NOAR) recruits individuals aged 16 years or older
at symptom onset, who have swelling of at least two joints persisting for at least
four weeks. Patients are notified to NOAR by primary care physicians or hospital rheumatologists
in the catchment area [18]. A subset of consecutive patients recruited between January 2004 and December 2008
by the main NOAR cohort were also enrolled into this CVD sub-study if they were 18
to 65 years old and assessed within 24 months of joint symptom onset. Informed consent
was obtained from patients and Norfolk Research Ethics Committees approval.

Manifestations of inflammatory polyarthritis

At inclusion into the NOAR cohort, patients were interviewed by a research nurse.
Current and previous medications for IP, as well as start and stop dates, were established.
Patients were considered to have been exposed to a therapy if they reported any current
or prior use. Fifty-one joints were assessed for the presence of swelling and tenderness.
Fasting blood was collected, separated and frozen at -80°C in Norfolk before being
transported to the Arthritis Research UK Epidemiology Unit in Manchester, UK, for
further analysis. A Hitachi (BMG Labtech LTD, Aylesbury, UK) 917/911 automated analyser
was used to determine C-reactive protein (CRP) concentration. The rheumatoid factor
(RF) was measured using a particle enhanced immunoturbidimetric assay where > 40 iU/ml
was considered positive for RF (Orion Diagnostica, BMG Labtech LTD, Aylesbury, UK).
Antibodies to citrullinated protein antigens (ACPA) were measured using the Axis-Shield
DIASTAT kit (Axis-Shield, Dundee, UK) where > 5 U/ml was considered positive for ACPA.
The 28-joint Disease Activity Score (DAS28) was calculated based on 28 tender joint
count, 28 swollen joint count, CRP and visual analogue scale (VAS) for general well-being
[19]. The UK version of the Health Assessment Questionnaire (HAQ) was completed by the
patient [20]. The 1987 American College of Rheumatology (ACR) classification criteria for RA were
applied [21].

Cardiovascular risk factors

Patients were classified as never smokers, previous smokers (if they had stopped smoking
prior to the interview) or current smokers. Measurement of height and weight was carried
out to calculate body mass index (BMI). Individuals were classified as being obese
if their BMI was ≥ 30 kg/m2. Diabetes was considered to be present if patients reported a physician diagnosis
of diabetes, if they were on treatment for diabetes, or if their fasting blood glucose
was ≥ 7.1 mmol/L on the day of assessment. Total cholesterol, high density lipoprotein
(HDL) and triglycerides were assayed on fresh fasting serum using CHOD-PAP, a homogenous
direct method (Abbott Diagnostics, Berkshire, UK) and GPO-PAP methods respectively
in Norfolk. LDL levels were mathematically derived from the total cholesterol and
HDL values.

Insulin resistance

Serum insulin levels were analysed using an ELISA kit from DRG diagnostics (Immunodiagnostic
Services, Boldon, UK) on fasting frozen serum samples in Manchester. Serum insulin
was measured by sensitive ELISA (Immunodiagnostic Systems Ltd, Boldon, UK). The kit
employs a monoclonal antibody to human insulin, which shows no cross-reactivity to
proinsulin. Insulin standards were calibrated against the World Health Organisation
(WHO) international reference preparation 66/304. The analytical sensitivity of the
assay was 1.76 mIU/L and intra and inter assay coefficients of variation were < 3.0%
and < 6.0% respectively. The manufacturer's reference range in apparently normal people
is 2 to 25 mIU/L. Insulin resistance was calculated using the Homeostasis Model Assessment,
a model which allows derivation of insulin resistance (HOMA-IR) and pancreatic beta
cell function (HOMA-B), calculated from fasting insulin/glucose pairs using homeostasis
model assessment software, HOMA2, downloaded from the Diabetes Trials Unit, University
of Oxford. This is an algorithm modified from the original by Jonathan Levy (Ref.
Levy JC, Matthews DR and Hermans MP. Correct Homeostasis Model Assessment (HOMA) Evaluation
uses the computer program Diabetes Care 1998, 21:2191-2192) IR was defined using the homeostatic model assessment (HOMA-IR): (Fasting
insulin μU × Fasting glucose mmol/ml)/22.5.

Patients with a HOMA-IR value of ≥ 2.29 were classified as having IR as recommended
in the literature [22].

Statistical analysis

The baseline characteristics of patients with normal IR levels and patients with high
IR levels were compared. For continuous variables, histograms were examined to ascertain
if the variables were normally distributed. A t-test and Mann-Whitney U tests were used accordingly to compare demographic and clinical
characteristics. Categorical variables were compared using the χ2 test. Linear and logistic regression analyses, with adjustment for age and gender,
were used to assess the association between the various traditional risk factors (TRFs)
for CVD and IP related parameters with HOMA-IR and IR respectively. Linear and logistic
regression was used to assess the association between RF and HOMA-IR and IR status
as a binary variable, respectively. This was repeated for ACPA. Four groups were identified
according to their serological status, that is: 1) negative for both RF and ACPA,
2) RF positive only, 3) ACPA positive only and 4) positive for both RF and ACPA. Linear
regression using these four categories as the independent variable was used to examine
the association between autoantibody status stratified into the above four categories
and IR. All analyses were repeated, adjusting for the presence of TRFs for CVD and
IP related parameters. The log likelihood test was used to analyse the degree of difference
between the four groups and their association with IR. All analyses were carried out
using the Stata 10 software package (Stata, College Station, TX, USA).

Results

Clinical characteristics of the cohort

We studied 196 patients, including 59 (30%) males, with a median (IQR) age and IP
symptom duration of 49 (40 to 57) years and 6.7 (4.6 to 10.7) months respectively.
Baseline characteristics are summarised in Table 1. Of note, 90 (47%) were RF positive, 66 (34%) were ACPA positive and 87 (44%) fulfilled
1987 ACR criteria for RA at baseline. The median (IQR) HOMA-IR was 2.7 (1.8 to 3.9)
in the entire cohort and 118 (60%) were insulin resistant (HOMA-IR ≥ 2.29). Patients
with IR had a higher prevalence of obesity, higher blood pressure and triglyceride
levels and lower HDL levels. A higher proportion of insulin resistant patients were
RF or ACPA positive (Table 1).

Table 1. Characteristics of 196 patients with recent onset IP and in those with and without
IR

Factors associated with insulin resistance

In an age and gender adjusted linear regression analysis, HOMA-IR was significantly
associated with a number of established cardiovascular and metabolic factors including
obesity, systolic and diastolic blood pressure, triglyceride levels and HDL. These
associations were also seen when we considered IR as a dichotomous outcome (Table
2).

HOMA-IR was associated with tender joint counts, and HAQ score (β-Coefficient (95%
CI); 0.029 (0.002, 0.056) and 0.709 (0.237, 1.182), respectively). In addition, patients
who were seropositive for RF or ACPA had a significantly higher HOMA-IR score (β-Coefficient
(95% CI); 0.924 (0.254, 1.594) and 1.051 (0.336, 1.767) respectively) and were more
likely to be insulin resistant (Table 2). The association between RF and ACPA status and HOMA-IR remained after adjustment
and/or removal of patients with known diabetes mellitus, those already taking DMARD
or steroid therapy, and after adjustment for other CVD risk factors and IP-related
factors examined (fully adjusted β-Coefficient (95% CI) for RF = 0.867 (0.204, 1.530)
and ACPA = 1.423 (0.701, 2.146) respectively).

Both RF and ACPA status were associated with HOMA-IR. In the small number of patients
positive for only RF or ACPA (15% and 3% respectively) there was no association with
HOMA-IR. However, there was a significant association between being positive for both
RF and ACPA and HOMA-IR (Table 3). Patients with both RF and ACPA had a significantly stronger association with HOMA-IR
than the RF positive group (log likelihood test (P = 0.0061) (Table 3).

Table 3. Associations between rheumatoid factor and anti-CCP antibodies with insulin resistance
alone and in combination with one another after serial adjustment

One hundred (51%) patients were seropositive for RF or ACPA. Examination of this subset
revealed similar associations with IR as was observed in the whole cohort including
a significant association between HOMA-IR and tender joint counts and HAQ score (β-Coefficient
(95% CI); 0.049 (0.004, 0.094) and 0.780 (0.071, 1.489), respectively) but not DAS28
score, CRP or swollen joint counts.

Discussion

In this cohort of patients with IP we have found a significant association between
serological status (RF and ACPA) and insulin resistance measured as HOMA-IR. This
association persists after adjustment for classic cardiovascular risk factors and
other IP-related factors. As far as we are aware, this is the first time this observation
has been noted in an early IP population.

A number of previous studies have examined IR in the context of established RA, usually
drawn from hospital cohorts. These studies have demonstrated increased IR in RA [7,23]. Most studies have demonstrated that insulin levels are associated with other metabolic
factors generally clustered within the Metabolic Syndrome [9,24]. The association between therapy (steroids in particular) and insulin levels has,
however, been controversial [23,25,26]. There is, however, evidence that IR may be related to inflammatory disease burden
[27]. This association has been supported by observations of reduced insulin resistance
following control of disease with anti-TNF therapy [15,16]. With regard to clinical outcomes, insulin resistance in RA has been associated with
both subclinical atherosclerosis and clinical cardiovascular disease [7,28,29]. Given that most study series of RA patients will have a very high percentage who
are seropositive, it may not be surprising that the association we have described
in our study has not been observed in these contexts. In addition, several previous
studies did not report ACPA status. Our previous studies have demonstrated that seropositivity
is an important prognostic factor in patients with IP and we have shown that it is
in this subgroup that there is a particular excess of CVD mortality [1,2,4].

In our cohort, IR was associated with the typical pattern of metabolic changes expected
from other population studies. Higher levels of IR are associated with obesity, fasting
blood glucose, blood pressure, triglycerides and low HDL. We also found a clear and
consistent association between seropositivity for RF and ACPA with IR, whether this
was measured across the range of HOMA-IR or if we treated IR as a categorical outcome.
With regard to other IP-related parameters, we did find that higher levels of HOMA-IR
were associated with tender joint counts and HAQ score at baseline; however, the lack
of association with DAS28 scores, swollen joint counts and acute phase reactants argues
against a strong influence of inflammatory disease burden in this cohort. Previous
general population studies have also shown that TNF-α and IL-6 are increased in insulin
resistant states [30,31]. TNF-α is known to interfere with both glycaemic sensing and insulin signalling,
thus impairing glucose handling [31]. The observation by others that TNF blockade reduces IR, would also support the hypothesis
that inflammatory disease burden contributes to IR in IP [32]. Our data, however, suggest that serological status may be a dominant factor determining
levels of IR in inflammatory polyarthritis patients.

While severe insulin resistance secondary to insulin receptor antibodies in the context
of type I diabetes mellitus is widely recognized [33], there are limited published data describing the association of insulin resistance
with other autoantibodies. RA and its related conditions overlap with other autoimmune
conditions and affect many organs. It is possible that the same triggers to autoimmune
RF and ACPA production also contribute to insulin receptor antibodies sufficient to
induce the degree of insulin resistance that we see in our study. We note a higher
level of insulin production and, therefore, islet cell destruction is unlikely to
be the mechanism by which this insulin resistance is induced.

We did not find any significant contribution of therapy to IR in our cohort. However,
in this early cohort only 22% of patients were exposed to steroids by the time of
the study and 54% of patients had been recently started on DMARD therapy. DMARDs may
of course reduce IR through anti-inflammatory effects, and this may also even be true
of low-dose steroid therapy [34]. We plan to follow this cohort to determine whether therapy does play a role in influencing
the metabolic status of these patients over time.

Most of the association between serological status and IR remains unexplained in our
fully adjusted models. We also note that the association was particularly strong in
those who are positive for both antibodies, although it should be pointed out that
this was the majority of seropositive patients. RF and ACPA are associated with the
presence of the shared epitope, and can be positive for many years prior to the onset
of arthritis. From a prognostic viewpoint, seropositivity also predicts future cardiovascular
mortality risk [35,36]. Seropositive patients are also more likely to develop extra-articular features such
as nodules, lung disease and vasculitis [37-39]. Other groups have found evidence for endothelial cell activation and dysfunction
in seropositive patients [40,41] and in RA patients who carry the shared epitope [42]. Insulin resistance, therefore, may be a consequence of early endothelial dysfunction
in seropositive patients. Insulin stimulates disposal of glucose from the circulation
into skeletal muscle and in eNOS knockout mice, capillary density is reduced and insulin
mediated glucose clearance is reduced by 40% [43]. Therefore, endothelial dysfunction is hypothesised to be a primary step in the development
of the insulin resistant state.

The development of RF and ACPA has also been associated with the shared epitope, and
with smoking status [1,44,45]. In addition, we have previously found that obesity is a risk factor for developing
IP [46]. An alternative explanation for the observations found is that smoking and obesity
contribute to both the IR and seropositivity in the population at risk of developing
IP. Although our study looked at patients early in the course of IP, ultimately this
question can only be answered in a pre-symptomatic population as we cannot accurately
determine which came first in this population.

There are several limitations which should be considered in this study. First, our
study is limited by its cross-sectional nature and longitudinal studies are required
to examine in closer detail the temporal and causal relationship between adiposity,
classic risk factors for CVD, inflammatory joint disease and therapy on the development
of IR. Conversely, by examining this question in a community-based, early IP population,
we have noted an important association between serological status and IR. Secondly,
the limited number of patients did not allow for further stratification of our results,
in particular with regard to the dose and cumulative exposure of oral steroid therapy
and its associations with IR. A further limitation is that we did not have the facilities
in a community-based study to undertake hyperinsulinaemic euglycaemic clamp experiments,
which are the 'gold standard' for ascertaining IR levels [22]. The HOMA-IR has, however, been validated as being an appropriate alternative from
a blood sample collected from our patients in their own home [47]. Multiple testing using both HOMA-IR as a continuous variable and IR as a binary
variable may be an issue. Whilst HOMA-IR may be considered more statistically valid,
the use of IR as a binary variable is more clinically useful and so both outcomes
are presented here, with results that were consistent using both approaches.

Conclusions

In conclusion, we have found that in an early IP population, IR is associated with
the presence of RF and ACPA, an effect that persists after adjustment for metabolic
and other IP-related factors. Insulin resistance is, therefore, detectable early in
the course of IP and may, in part, explain the excess CVD risk observed in seropositive
patients. A better understanding of the mechanisms underlying this observation may
shed light on the pathogenesis of accelerated atherosclerosis in IP patients.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

HM contributed to the design, data collection and analysis for this paper and wrote
the manuscript. TF is the previous project lead and was involved in coordinating and
supervising data collection, analysis and manuscript writing. SV is the project lead
and coordinated and supervised data collection, analysis and manuscript writing. AY
was the laboratory lead in analysing the serum insulin levels. DB is the Clinical
Manager in charge of patient recruitment, consent, follow-up and data collection.
TM is a Consultant Rheumatologist and is involved in patient recruitment for the study.
ML is a Senior Lecturer in Statistics and contributed to study design and analysis.
DS is the founder and principal investigator on the NOAR study. IB is the co-principal
investigator and was instrumentally involved in study design, analysis and manuscript
preparation. All authors have read and approved the manuscript for publication.

Authors' information

HM is a Clinical Research Fellow at the Arthritis Research UK Epidemiology Unit at
the University of Manchester.

Acknowledgements

We acknowledge nursing support from Jacquie Chipping, Rowena Pavey, Alison Lincoln,
Jenny Doyle, Lynn Massingham; and administrative support from Caroline Heasley. This
study was funded by the Arthritis Research UK (Grant No. 17552). Professors Bruce
and Symmons are supported by the Manchester Academic Health Sciences Centre.